White blood cells called T lymphocytes play critical roles in immune defense against viruses, bacteria, fungi, protozoa, and cancer cells. They are also involved in allergies/asthma due to the development of an unwanted or excessive type of immune response to substances (antigens) in our environment and in autoimmune diseases. Because T cells respond to foreign substances (antigens) in the form of peptide- major histocompatibility complex (MHC) molecule complexes on cell surfaces, we wish to know how such complexes interact with specific receptors to evoke the effector activities of mature T cells in the body, as well as regulate their growth, inactivation, or death. In particular, we want to understand in molecular detail the protein-protein interactions that turn recognition of antigen by T cells into signals guiding the normal survival and effector functions of these cells, how variations in these recognition and signaling events leads to desirable versus undesirable forms of immunity, and how we can manipulate these events to augment useful immune responses and inhibit damaging ones. Our studies currently focus on biochemical regulatory pathways that help T-cells discriminate between self- and foreign peptide:MHC molecule complexes, on the role of self-recognition in the sensitivity of T-cells to antigen on presenting cells of the types studied in LI545, and on the explicit mathematical modeling of these signaling processes in eukaryotic cells. We previously reported our analysis of the molecular details of two novel regulatory pathways controlling early signaling by the T-cell receptor (SHP-1 phosphatase dependent negative control and MAPK mediated positive control). This past year we completed a first full mathematical / computer description of T cell receptor (TCR) signaling that incorporates these novel regulatory pathways. Simulation results from this model indicate that these feedback controls play a crucial role in creating a sharp transition from non-agonist to agonist behavior for ligands with varying affinities for the receptor. Several other predictions of the model that involve antagonist function, how ligand discrimination changes with the activation state of a T cell, and the rate of ERK activation were all validated by experiment. We have extended our studies showing that recently activated T cells acquire overt reactivity to self-antigens, which we believe helps expand clonal precursors during the period of antigen limitation early in an infectious process. We have determined that a widely-expressed protein is synthesized in T cells with a slight delay after Initiation of signaling and that it functions to produce attenuated but long-lasting signals required for clonal expansion prior to development of effector function. In the course of these studies, we also discovered that several hours after TCR stimulation most T cells begin to express a negative regulatory transcription factor that limits the extent and duration of cytokine production. Whether this occurs or not is related to the quality and amount of TCR and costimulatory signaling the T cell receives and involves regulation at the level of protein degradation through indirect control of a protease. Our preliminary data suggest that this negative factor expression is key to limiting autoimmune responses, while promoting useful immunity to foreign antigens.